Many clinically important classes of antibiotics, including macrolides (azithromycin, telithromycin), tetracyclines, aminoglycosides (gentamycin), and more recently oxazolidinones (linezolid) target the bacterial ribosome, which is essential for intracellular protein synthesis. Use of these and other antibiotics against pathogenic microorganisms inevitably leads to development of bacterial resistance, an increasing problem within the clinical setting. There is thus a continued need to develop new antibiotics, effective against multi-drug resistant bacteria, and for approaches which can retard the inevitable development of resistance to these. The ribosome represents one of the richest and most effective targets for development of new antibiotics. Such drug development efforts can build upon a molecular level understanding of the ribosomal interactions with chemically distinct architectures, structural requirements for other aspects of biologically activity (including cellular uptake) and likely mechanisms for development of resistance. Hygromycin A (HygA), an antibiotic isolated from Streptomyces hygroscopicus and is active against both Gram-positive and Gram-negative bacteria and a mechanism of action which involves a distinct but enigmatic binding to the ribosome. HygA also has a chemical architecture which differs significantly from other antibiotics which target the ribosome. For these reasons HygA is of interest for potential development into new class of clinically-useful antibiotics. The long term project objective is to build a comprehensive understanding of hygromycin A (HygA) activity, biosynthesis, export, resistance and regulation (ABERR). This project period has 5 specific aims which will be pursed using a multi-interdisciplinary and collaborative approach with genetic, biochemical and structural studies. Aim 1. Obtain a detailed understanding of the enzymology of key and novel steps in the HygA biosynthetic process. Aim 2. Elucidate how HygA, biosynthetic pathway intermediates and shunt products, bind to ribosomes. Aim 3. Determine mechanisms by which the final product HygA is excreted from the cell. Aim 4. Evaluate the relative roles of covalent modification of both the antibiotic and the S. hygroscopicus ribosome in conferring resistance to HygA and related biosynthetic products. Aim 5. Determine how HygA ABERR processes are regulated, and to what extent are they coordinated. PUBLIC HEALTH RELEVANCE: There is continued need for new antibiotics effective against multi-drug resistant bacteria, and for approaches which can retard the inevitable development of resistance to these. Actinomycetes are soil bacteria and serve both as a source for the majority of antibiotics used clinically to treat bacterial infections and a repository for resistance mechanisms to these. This study will evaluate biological activity and resistance mechanisms associated with the natural product hygromycin A and related compounds and may ultimately lead to new therapies for treatment of bacterial infections.